Apparatus and method for preparative scale purification of nucleic acids

ABSTRACT

Apparatus and methods are described for pharmaceutical grade manufacture extrachromosomal nucleic acids from cell lysates using flotation to separate and eliminate undesired insoluble cellular debris including chromosomal DNA from the lysates. A gas is introduced to controllably generate bubbles that reduce the density of the cell debris and create a buoyant flocculent phase that can be readily separated from, and thus provide, a substantially clarified fluid lysate phase that is enriched in extrachromosomal DNA but substantially depleted of cellular proteins and chromosomal DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application 60/410,617, filedSep. 13, 2002, incorporated herein by reference.

TECHNICAL FIELD

The invention relates to methods for purifying nucleic acids. Theinvention relates in particular to methods for preparing pharmaceuticalquality plasmid DNA at preparative scale.

BACKGROUND OF THE INVENTION

Since the advent of recombinant DNA, methods have been developed andimproved for the purification of DNA and RNA to further molecularbiology research. While these methods have allowed considerable study ofnucleic acids in research environments, methods for preparative scaleproduction of plasmid DNA sufficient in quantity and quality forclinical use have been problematic and continue to represent an unmetneed.

Gene therapy involves the introduction of nucleic acid into a patient'scells, which, when expressed, provide a therapeutic benefit to thepatient. Examples include the introduction of an exogenous, functionalgene to correct a genetic defect in a patient carrying a defective geneor to compensate for a gene that is not expressed at sufficient levels.Other examples include the introduction of mutant genes, antisensesequences or ribozymes to block a genetic function, e.g., in thetreatment of viral infections or cancer.

For any application in which nucleic acid is introduced into a human oranimal in a therapeutic context, there is a need to produce highlypurified, pharmaceutical grade nucleic acid. Such purified nucleic acidmust meet drug quality standards of safety, potency and efficacy. Inaddition, it is desirable to have a scaleable process that can be usedto produce multiple gram quantities of DNA. Thus, it is desirable tohave a process for producing highly pure nucleic acid that does notrequire toxic chemicals, known mutagens, organic solvents, or otherreagents that would compromise the safety or efficacy of the resultingnucleic acid, or make scale-up difficult or impractical. It is alsodesirable to prepare nucleic acids free from contaminating endotoxins,which if administered to a patient could elicit a toxic response.Removal of contaminating endotoxins is particularly important whereplasmid or bacteriophage DNA is purified from gram-negative bacterialsources that have high levels of endotoxins as an integral component ofthe outer cell membrane.

Preparative scale plasmid manufacturing most commonly involves alkalinelysis of bacterial cells containing extrachromosomal DNA of interestsuch as plasmid or phage DNA. Alkaline lysis was first developed byBirnboim and Doly, Nucleic Acids Res 1979; 7(6):1513-23, as a screeningmethod for recombinant plasmids. As Birnboim and Doly found, alkalinelysis of bacteria effects release of intracellular plasmid DNA togetherwith selective denaturation of chromosomal DNA that renatures uponneutralization to form an insoluble “clot” together with cellulardebris. The lysate from an alkaline lysis process, such as for examplethe method of Birnboim and Doly, usually consists of a slurry ofprecipitated or flocculated debris suspended in a golden yellow coloredliquid. The plasmid DNA remains predominantly in the liquid portion ofthe neutralized solution. To obtain the liquid containing desiredextrachromosomal polynucleotides, the debris has to be removed from theslurry without shearing of either the precipitated chromosomal DNAdebris or the extrachromosomal polynucleotide products. As in theminipreparative technique of Birnboim and Doly, centrifugation is themost commonly used method to isolate the liquid from the solidprecipitates. On a preparative scale, various means have been employedto clarify the alkaline lysate including centrifugation, sedimentation,and filtration. Centrifugation has been commonly employed. (Bussey etal., U.S. Pat. No. 6,011,148; Butler et al., U.S. Pat. No. 6,313,285).Filtration means have included bag filtration (Thatcher et al., U.S.Pat. No. 5,981,735), depth filtration (Mittelstaedt and Hsu, U.S. Pat.No. 6,268,492) and filtration with diatomaceous earth (Horn et al., U.S.Pat. No. 5,576,196).

While continuous-flow centrifugation can be efficient for theseparation, the shearing motion can result in irreversible damage to theplasmid. On the other hand, batch centrifugation involves completecontainment of the lysate inside centrifuge bottles, which prevents thedegradation during centrifugation. However, scaling up the batchcentrifugation process has been constrained by the lack of commercialavailability of larger centrifuges. Also, having multiple centrifugesmay not be feasible, due to the prohibitive expense. Thus, as the scaleof the production run increases, the volume of material makes thetraditional centrifugation, sedimentation, and filtration means tooinefficient, time consuming and/or expensive. A more efficient method ofremoving the precipitated debris is therefore needed.

The invention described provides a novel method and apparatus forclarification of cell lysates that is rapid, highly efficient,relatively inexpensive and conducive to automation.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method for purifying nucleicacids from lysed cells that is suitable for preparative scalemanufacture. The method and apparatus of the invention providesautomatable manufacturing of high quality extrachromosomal nucleicacids, particularly plasmid and phage DNA, from bacterial cells.Extrachromosomal nucleic acids prepared according to the presentinvention are suitable for clinical use.

The invention provides novel methods and apparatus to separate celldebris precipitates from the liquid phase of cell lysates. Inparticular, the methods and apparatus of the invention are suitable forgenerating a clarified bacterial lysate using flotation to separate andeliminate undesired insoluble cellular debris including chromosomal DNA.According to the invention, gas such as for example, air or nitrogen, iscontrollably introduced into a lysis process in order to provide amotive force for phase separation by generating a buoyant flocculent orprecipitate phase containing cellular debris that is readily separatedfrom an underlying clarified liquid phase containing extrachromosomalnucleic acids.

In one embodiment, the gas is introduced to controllably generatebubbles that reduce the density of the cell debris and create a buoyantflocculent phase that can be readily separated from, and thus provide, asubstantially clarified fluid lysate phase that is enriched in plasmidDNA but substantially depleted of cellular proteins and chromosomal DNA.In one embodiment, the bubbles are controllably introduced and theresultant buoyant flocculent phase is allowed to coalesce and stabilizewithin a defined period of time, thus permitting defined manufacturingprocess parameters.

In one embodiment, the gas is controllably introduced through a devicethat generates gas bubbles of defined size. In one such embodiment,recovery of liquid lysate and the stability of the floating cell debrisis improved by adding the gas through an air or “sparge” stone.

In one embodiment, the methods involve a continuous flow in-line processincluding use of static mixers to mix the cells with a lysis solution toprovide controlled, gentle mixing of the cells with the lysis solution.Static mixers are further used to mix the resulting lysis mixture with aprecipitation solution to separate out cell debris and othercontaminants, including-chromosomal DNA.

Liquid phase recovery is maximized by optimizing the flow rates of bothair and liquid. In a one embodiment, the recommended conditions forflotation, plasmid yield, and lysate quality through an in-line lysisapparatus using an alkaline lysis procedure were found to be 0.3 ft/slinear velocity with 12% air introduced at the initiation of lysisthrough a stainless steel sparge stone having about 2 micron holes.

In further embodiments, the clarified lysate is further purified.Optionally a series of filters including depth filters and filtershaving charge characteristics sufficient to remove certain contaminantsare employed. In one embodiment, the clarified lysate of the presentinvention is passed through depth filters of decreasing pore size, forexample an about 8 to about 10 micron depth filter followed in serieswith an about 2 micron depth filter followed by passage through glassfiber and nylon filters to provide a filtered clarified lysate.

In a further embodiments, the filtered clarified lysate is furtherpurified by ion exchange chromatography to remove residual impuritiesincluding cellular proteins, chromosomal DNA, RNA and endotoxins.

The methods of the invention are sufficient to provide a purified DNAsolution in a high volume process that does not require a centrifugationstep subsequent to cell lysis. The process further does not requirecomplex purification steps (e.g., ultrafiltration) prior to ion exchangechromatography. Optionally the ion exchange-purified material is furthersubject to an ultrafiltration/diafiltration step.

When desired, the methods can be readily automated according towell-known methods by including appropriate computer controls of stepsin the process to ensure desired results. The invention also providesparticular conditions by which the methods can be used to prepareextrachromosomal polynucleotide drug products in an automated manner.

DESCRIPTION THE DRAWINGS

FIG. 1 illustrates a sparge stone assembly according to one embodimentof the present invention.

FIG. 2 represents several examples of locations tested for introductionof gas into an inline lysis apparatus and process.

FIG. 3 illustrates the relative placement of a sparge stone in anin-line lysis apparatus according to one embodiment of the presentinvention.

FIG. 4 illustrates a schematic diagram of an in-line bacterial celllysis apparatus in accordance with the present invention.

FIGS. 5A and B represent the relative effects of different quantities ofgas on the ultimate volume of a resultant liquid phase after separationdepending on whether the gas is introduced at the beginning of the lysisprocedure compared with entry of gas at the time the precipitationbuffer is added. In FIG. 5A the gas is introduced essentially togetherwith the cell lysis buffer. In FIG. 5B the gas is introduced essentiallytogether with the precipitation buffer.

FIG. 6 represents the relative lysate recovery volume after 2 hours ofresting flotation depending on different volumes of gas added through a“T” junction at different fluid flow rates.

FIG. 7 represents the relative lysate recovery volume after 1 and 2hours of resting flotation depending the relative effects of differentvolumes of gas added through a sparge stone.

FIG. 8 depicts the effects of linear velocity at different percentageair volumes on plasmid yield.

FIG. 9 depicts the effects of linear velocity at different percentageair volumes on residual endotoxin levels.

FIG. 10 depicts the effects of linear velocity at different percentageair volumes on residual chromosomal DNA content.

FIG. 11 is a general process flow chart for plasmid purificationaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be utilized in a highly streamlined processfor the purification of high quality pharmaceutical gradepolynucleotides at a preparative scale. The process minimizes complex orexpensive purification steps, thus minimizing cost and allowing anincrease in throughput. It is also an advantage of the invention thatthe process is readily automated and minimizes the use of apparatus thatmay shear the chromosomal DNA, thus avoiding potential contamination ofthe extrachromosomal nucleic acid preparation. The process isparticularly suitable for providing pharmaceutical grade plasmid andphage DNA at commercial scale from bacterial cell lysates.

The general process sequence for producing pharmaceutical grade plasmidDNA from bacterial cells is depicted in FIG. 11 as follows: 1)inoculation from a master cell bank; 2) fermentation; 3) harvest andproduction of a cell paste; 4) cell lysis and clarification of thelysate; 5) ion exchange chromatography; 6)ultrafiltration/diafiltration; and 7) final filtration to produce a bulkdrug substance.

In one embodiment, an apparatus is provided for producing a clarifiedbacterial lysate according to the following steps: 1) lysis of abacterial cell suspension; 2) controlled introduction of a gassufficient to float a subsequent precipitate/flocculent; 3) introductionof an agent that effects precipitation/flocculation of cell debris fromthe lysed bacterial cells; and 4) separation of theprecipitate/flocculent resulting in isolation of a clarified lysate.

For purposes of the present invention, the term “precipitate” means nolonger soluble or in solution. As such, the terms “precipitate” and“flocculent” are used interchangeably herein to refer to the formationof essentially insoluble clumps of cellular debris, including cell wallconstituents, chromosomal DNA and protein, that form as a consequence ofthe lysis of live cells and chemical treatment of such lysate. Thepresent inventors found that portions of the “flocculent” tended tofloat and devised apparatus and methods that result in flotation of amajority of the visual precipitate thus resulting in highly effectivephase separation from a resulting clear fluid phase containing theextrachromosomal nucleic acid product of the fermentation.

In one embodiment, gas in the form of compressed air, or alternativelynitrogen or other pharmaceutically acceptable gas or mixture of gases,is controllably introduced into an aqueous lysis mixture in order tocontrollably form bubbles that effect flotation of cellular debrisprecipitates resulting from chemical treatment of the lysate. Gas can beintroduced into the process through any means that is amenable tocontrol of the volume of gas introduced and through which bubbles can becontrollably formed in a lysis solution. Thus, it is desirable to haveflow control means for the introduction of the gas. Gas can beintroduced through any port including for example “T” junctions,perforated rigid or flexible tubing, or injection ports and may furtherinclude means for the generation of small bubbles such as, for example,air or “sparge” stones or perforated filter disks.

In a preferred embodiment, the size of gas bubble produced is controlledby introducing the gas through an aperture having a multiplicity ofsmall holes, preferably of relatively uniform size. In this embodiment,any apparatus able to generate small bubbles, such as for examplesintered glass disks, disc filters or aeration stones or sparge stonesformed of glass, plastic, or metal, may be employed. For purposes of thepresent invention the term “sparge stone” refers to any perforated meansthrough which gas can be introduced into the liquid lysis milieu andform small bubbles. In one embodiment, a sparge stone is placed in theinput line for an alkaline lysis buffer. The sparge stone should becomposed of a material that is non reactive with the lysis milieu. Theapparatus may be composed of a material that can be cleaned and reusedor may be disposable.

In one embodiment, the gas is introduced into the lysis milieu through astainless steel sparge stone. Inert metal sparge stones may be usedrepeatedly and may be cleaned by acid/base washes and sterilized byautoclaving. In a preferred embodiment, the sparge stone is orientedvertically in the fluid flow path in such a way that the bubbles formedare allowed to rise directly up into the fluid stream.

In one embodiment, a sparge stone is employed having small holes orpores of a relatively uniform diameter. In one embodiment the pores havean average diameter of less than about 5 microns in diameter. In onepreferred embodiment, a stainless steel sparge stone having uniformpores of approximately 2 microns in diameter is employed resulting inthe formation of uniform small bubbles and an overall volume of gas ofapproximately 12% of the total volume. Large bubbles, if desired, mayrequire adjustment to the use of larger gas volumes. Larger bubbles mayrequire a faster flow rate in order to accommodate the volume of gas andresult in greater trapping of fluid in the floating layer ultimatelyformed.

In one embodiment, the unclarified lysate is allowed to separate fromthe cell debris precipitate/flocculent, including introduced bubbles,for approximately 1 to 2 hours at ambient temperature. The precipitatefloats to the top of the tank due to the air bubbles introduced into themixture. Typically, the controlled introduction of gas into theprocedure permits in immediate phase separation of a buoyant flocculentphase following addition of the precipitation buffer. However, in oneembodiment the mixture is allowed to rest in a tank for approximately 1to 2 hours in order for the buoyant flocculent to coalesce and solidifyprior to removal of the clear clarified lysate phase from under thebuoyant precipitate/flocculent phase. The configuration of the lysatetank is modified depending on the volume of material to be separated.Thus, even small volumes may be effectively separated using a tall,narrow tank. Surprisingly, in large volume lysis the separation effectedthrough flotation of the precipitate allows for isolation of a clarifiedlysate in 1 to 2 hours that is superior to that obtained with over 8hours of centrifugation.

In one embodiment, a continuous flow in-line apparatus is provided forproducing a clarified bacterial lysate by conducting the following stepsin accordance with FIG. 4: 1) in-line chemical lysis of a bacterial cellsuspension in conjunction with controlled introduction of a gassufficient to float a subsequent precipitate/flocculent; 2) in-lineprecipitation/flocculation of cell debris from the lysed bacterialcells; and 3) separation of the floating precipitate/flocculent phasefrom a resulting clarified lysate phase. FIG. 4 presents an embodimentincluding a step of adjusting the pH of the lysate before separation ofthe precipitate/flocculent to protect the DNA from depurination and togenerate a clarified or filtered lysate that may be directly applied toan ion exchange step. Alternatively, if the lysis and precipitationconditions result in an essentially neutral pH, addition of a further pHadjustment buffer may be avoided. Thus, in one embodiment the pHadjustment step is considered optional depending on thelysis/precipitation conditions.

In one embodiment the process involves a lysis procedure including asurfactant that enhances the formation of a buoyant flocculent uponaddition of a compound that induces precipitation/flocculation ofchromosomal DNA and cellular debris from the lysed cells.

In one embodiment, apparatus and methods are provided for alkaline lysisin which bacterial cells from a previously collected cell paste areresuspended in 25 mM Tris, 10 mM Na₂EDTA, 83 mM Dextrose pH 8.50 at aratio of 5 liters per kilogram of cell paste. The resuspended cells arethen introduced into a in-line continuous flow apparatus wherein theyare lysed with an alkaline lysis buffer (0.2M NaOH, 1% SDS;approximately 10 liters per original kilogram (kg) of cell paste) inconjunction with controlled introduction of a gas sufficient to float asubsequent precipitate/flocculent, followed by in-lineprecipitation/flocculation using 3 M potassium acetate/2M acetic acid,pH 5 (5 liters per kg of cell paste). A further pH adjustment buffer(2.5 M Tris, pH 8.5, 4-5 liters per kilogram of paste) is added in-lineprior to flotation of the precipitate/flocculent.

In one embodiment, RNase is added to digest RNA released by the cellsafter lysis. For example, in one embodiment 5 mL of RNase A per kg ofcell paste (approximately 15,000-24,000 Kunitz units/kg) is added todigest RNA and the cell suspension is mixed for 1-2 hours at ambienttemperature using bow-tie impeller prior to in-line lysis.Alternatively, methods may be employed to reduce or eliminate thebacterial RNA without using RNase. For example, prolongation of thealkaline lysis step for 4 to 24 hours together with the addition oflysozyme has been employed to reduce the levels of RNase. (Butler etal., U.S. Pat. No. 6,313,285). Alternately, residual RNA and chromosomalDNA can be reduced by ammonium sulfate precipitation following alkalinelysis, precipitation, and clarification. (McNeilly et al., U.S. Pat. No.6,214,586). Other methods that may be employed include the use ofbacterial strains in which a gene encoding RNase is stably introducedinto the bacterial host genome or transiently expressed via a plasmid.Expression of the RNase gene is induced near the end of fermentation inorder to produce a local source of the RNase enzyme.

“Extrachromosomal nucleic acids” include DNA, RNA and chimeric DNA/RNAmolecules that do not constitute the primary genome of eukaryotic orprokaryotic host cells. Nucleic acids that may be purified includeribosomal RNA, mRNA, snRNAs, tRNA, plasmid DNA, viral RNA or DNA. Thepresent method is particularly suitable for the purification ofextrachromosomal phage and plasmid DNAs from prokaryotic cells. Ofparticular interest are plasmid DNAs encoding therapeutic genes. By“therapeutic genes” is intended to include functional genes or genefragments which can be expressed in a suitable host cell to complement adefective or under-expressed gene in the host cell, as well as genes orgene fragments that, when expressed, inhibit or suppress the function ofa gene in the host cell including, e.g., antisense sequences, ribozymes,transdominant inhibitors, and the like. Plasmid DNA isolated fromprokaryotic cells include naturally occurring plasmids as well asrecombinant plasmids encoding a gene of interest including, e.g., markergenes or therapeutic genes.

The term “static mixer” refers to any flow-through device that providesenough contact time between two or more liquids to allow substantiallycomplete mixing of the liquids. In the methods of the invention, staticmixers are used to mix certain solutions, particularly where gentle andcomplete mixing is desired. Typically, static mixers contain an internalhelical structure which allows the liquids to come in contact in anopposing rotational flow and causes them to mix in a turbulent orlaminar flow. Such mixers are described, for instance, in U.S. Pat. No.3,286,922. Such devices are useful, for instance, in isolating plasmidDNA from lysed bacterial cells, or for precipitating cell debris,proteins, and chromosomal DNA after lysis, as described, for instance,in Christopher et al., U.S. Pat. No. 5,837,529. During these procedures,the mixing should be complete to maximize recovery and should berelatively quick to maintain DNA integrity. If mixing is too vigorous,however, genomic DNA can be sheared and may contaminate theextrachromosomal polynucleotide preparation. Static mixers areadvantageous in these applications because substantially complete mixingcan be obtained while minimizing shear of genomic DNA. In addition,lysis is typically carried out in caustic solutions such as alkali,which can affect the quality of the final preparation. Since staticmixers allow continuous flow, the time in contact with these solutionscan be carefully controlled.

The degree of mixing is controlled by varying the linear velocity orflow rate of the solution through the mixer, the type of mixer used, thediameter of the mixer, and the number of elements in the mixer. Forinstance, in the preparation of plasmid DNA from bacterial cells alaminar flow static mixing environment is preferred such can be obtainedat certain flow rates with commercially available static mixers such asfor example KENICS brand sanitary mixers available from Chemineer, Inc.The linear velocity used depends on the manufacturer and type of mixer;this controls the Reynolds number achieved and how gentle the mixing is.

A linear velocity of 0.3 to 1.1 feet per second gives acceptable productquality when using a 2″ diameter, 36-element, laminar flow static mixer(obtainable for example by placing three 12 element static mixersin-line, i.e. KENICS 2 KMR-SAN 12 sanitary mixers, each individual 12element mixer having an overall length of 38 inches) with an overalllength of about 9 feet corresponding to a Reynolds number from 14 to 53(calculating the Reynolds based on a 2″ diameter (1⅞″ i.d.), assumingviscosity=300 cp.). This linear velocity range permits sufficient mixingto thoroughly lyse the cells and yet not be so high that genomic DNA issheared to a size that is problematic in later purification steps. At a0.7 feet per second linear velocity the flow rate in a 2″ diameter mixeris typically ˜22 liters per minute. The flow rate out of the 1^(st)mixer will be 22.8 L/min at the indicated linear velocity and size ofstatic mixer. The flow rate would be 30 L/min out of the 2^(nd) mixerdue to the increase in volume.

The term “continuous flow” or “continuous flow in-line fluid path”refers to an apparatus and system in which an apparatus provides thecapability to operate a constant flow process in a contained closed pathwithout discontinuous interruption. It is an advantage of the presentinvention that cell lysis, precipitation and clarification can beperformed in a continuous, automated process by use of static mixerswith appropriate adjustment of flow rates. The flow rate must besufficient to achieve adequate lysis, precipitation and neutralization,but not resulting in shearing of genomic DNA. Appropriate sizing of thestatic mixers, pumps, flocculent separation apparatus, and selection offlow rate will allow continuous operation of the process, whilemaximizing yield and quality. Preferably, the process is automated toensure reproducibility.

The term “precipitation buffer” is used to describe a solution that isused to precipitate proteins, chromosomal DNA and cell debris. Whenreferring to an alkaline lysis procedure, the precipitation buffer mayalso be termed a neutralization buffer as the precipitation occurs as aresult of neutralization of the alkaline lysate. Typically, for analkaline lysis utilizing 0.2 M NaOH and 1% SDS, the precipitationsolution will contain potassium acetate. A suitable precipitatingsolution is 3M potassium acetate, adjusted to pH 5.5, with 2M aceticacid (˜5M acetate final). When utilizing a static mixer, linearvelocities are used that ensure sufficient mixing to thoroughlyprecipitate the proteins and cellular debris and yet are not so highthat genomic DNA is sheared to a size that is problematic in laterpurification steps. Typically, approximately 4.5 liters of precipitatingsolution are used per kg of cell paste.

The term “pH adjustment buffer” is used to describe a solution that isused to raise the pH of cell lysate after addition of the precipitationsolution in order to minimize acid catalyzed de-purination of the DNAand to condition the material for binding onto the anion exchangecolumn, e.g., a pH is the range of 6 to 9, preferably from 7 to 8.5. Auseful buffer solution for this purpose is 2.5 M Tris (˜pH 11). In oneembodiment, the pH adjustment solution is added prior to separation ofthe buoyant flocculent in order to avoid disruption of the flocculent.

In a further embodiment, an apparatus and system is provided forproducing a clarified bacterial lysate in accordance with the followingsteps: 1) lysis of a bacterial cell suspension in conjunction withcontrolled introduction of a gas sufficient to float a subsequentprecipitate/flocculent; 2) introduction of an agent that effectsprecipitation/flocculation of cell debris from the lysed bacterialcells; 3) separation of the floating precipitate/flocculent phase from aresulting clarified lysate phase; 4) filtration of the clarified lysate;5) ion exchange chromatography of the filtered lysate; 6)ultrafiltration/diafiltration of the ion exchange eluate; and 7) finalfiltration to produce a bulk drug substance.

The following examples illustrate certain aspects of the above-describedmethod and advantageous results. The following examples are shown by wayof illustration and not by way of limitation.

EXAMPLE 1

In one embodiment, bacterial cells are lysed by contact with a stronglyalkaline solution in the presence of a detergent. A typical alkalinelysis solution is 0.2N sodium hydroxide (NaOH) with 1% sodium dodecylsulfate (SDS). The classical alkaline lysis procedure of Birnboim andDoly involved lysis of bacteria on a mini-preparative scale in whichalkaline conditions effected selective denaturation of chromosomal DNAthat renatured upon neutralization and, together-with cellular proteinsand debris, formed an insoluble “clot” that could be removed bycentrifugation. The neutralization step has been also termed“precipitation” for the formation of the insoluble “precipitate.” Theplasmid DNA remains in the fluid phase of the “neutralized” solution. Ona preparative scale, various means have been employed to clarify thealkaline lysate including centrifugation, sedimentation, and filtration.Filtration means have included depth filtration (U.S. Pat. No.6,268,492) and filtration with diatomaceous earth (U.S. Pat. No.5,576,196). However, as the scale of the production run increases, thevolume of material makes the traditional centrifugation, sedimentation,and filtration means too inefficient, time consuming or expensive.Furthermore, the alkaline lysis “precipitate” might be better describedas a “flocculent” in which insoluble material is produced but does notnecessarily rapidly settle out of solution.

In one determination of the density of constituents of the lysate by thepresent inventors, the density of the cell debris was determined to beapproximately 1.15 g/mL while the liquid phase was 1.07 g/mL. Based onthe difference between the specific gravity of these two phases, celldebris should fall out of solution. However, in practice this is notalways the case. Several different conditions of lysates coming out ofthe static mixers have included those having both floating and sinkingprecipitates, those having largely suspended precipitates and thosehaving a large component of floating “precipitate.”

Regardless of the initial appearance of the lysate exiting the staticmixers, the precipitates can typically be induced to form a pellet withsufficient centrifugation. However, prolonged centrifugation may berequired for clarification of large preparative scale volumes and it wasobserved that such centrifugation could result in shearing of thechromosomal DNA and contamination of the plasmid DNA with fragments ofchromosomal DNA.

Upon close visual examination, it was found that small air bubbles arepresent in the solid precipitates that initially float. Based on thisobservation, a theory was developed that the air trapped in the celldebris may be utilized to provide a buoyancy force needed to counter thehigher density of the precipitates. According to this theory, the celldebris could be substantially suspended, or floated, in the lysate if acritical amount of gas could be introduced.

Thus, in development of the present invention, efforts were undertakento devise new methods and apparatus for separating the precipitate basedon inducing the precipitate to float instead of attempting to remove theprecipitate by centrifugation or various filtration means. It wassurprisingly found that controlled introduction of air into the lysismilieu was able to permit flotation of virtually the entireprecipitate/flocculent resulting in high yield recovery of a highquality substantially clarified lysate.

Based on visual observation, most air bubbles that formed themeta-stable cell-debris/air complexes were less than 1 mm in diameter.It was hoped that the efficiency of the formation of such complexescould be improved with optimized air bubble production in the context ofthe entire lysis procedure.

EXAMPLE 2

Initial Testing: Location of air introduction: During lysis using thestatic mixer assembly, air was introduced via a separate peristalticpump into a ⅜″ i.d. static mixer arrangement as diagrammaticallydepicted in FIG. 2. Inside each of Static Mixers 210 and 211 are 36alternating twisted elements that facilitate better mixing (each ofStatic Mixers 210 and 211 are formed by connecting in series 3individual 12 element mixers: ½ inch o.d. KENICS ½ KMR-SAN 12 sanitarymixers obtainable from Chemineer, Inc.). The flow rates for cellre-suspension 23 (E. coli transformed with plasmid pMB290, OD 150), celllysis buffer 24 (0.2N NaOH; 1% SDS), precipitation buffer 27 (3M KOAc;2M HOAc) and neutralization buffer 28 (2.5M Tris-base) were 370, 620,310, and 265 mL/min, respectively. The air was introduced at twoalternative locations 5 or 6 via a “T” connection into the static mixerassembly. For each inlet, three settings (600, 900, 1800 mL/min) of airwere tested. The separation between the liquid (clarified lysate) andsolid (cell debris) phases in lysate 29 was recorded as a function oftime. Cell lysis with no air introduction was also run as a control.Table 1 below indicates the tested flow rates in ml/minute for the cellresuspension, and cell lysis precipitation and neutralization buffers.

TABLE 1 Liquid Flow Rates for ⅜″ Static Mixers Linear Velocity in StaticMixer #1 Flow Rates in mL/min 0.76 ft/s 1.14 ft/s 1.37 ft/s CellRe-suspension 370 560 670 Cell Lysis Buffer 620 930 1120 CellPrecipitation Buffer 310 465 560 Neutralization Buffer 265 400 480

Determination of Location for Introduction of Air: A total of sixlysates were generated from the static mixers, with air introduced ateither inlet. All six samples were found to contain floatingprecipitates. However, the degree of compaction of the precipitates wasnot identical for all samples. During the first hour, the interfacecontinued to rise up to 24-49%. After 16 hours, the liquid phaseoccupied 34-58%, depending on the airflow rate and location of theinlet. The control sample was found to be a suspension of precipitatesthat did not clarify by phase separation. The progress of precipitatecompaction over time is summarized in FIGS. 5A & 5B.

In FIG. 5A, air was delivered through a “T” located prior to the firststatic mixer in the flow path, as depicted at position 5 in FIG. 2. InFIG. 5B, air was delivered through a “T” located prior to the secondstatic mixer in the flow path, as depicted at position 6 in FIG. 2. Thepercentages shown on FIGS. 5A and 5B represent a ratio of the flow rateof air to the combined flow rates of all lysate constituents includingthe flow rates for the cell re-suspension (370 ml/min), cell lysisbuffer (620 mL/min), cell precipitation buffer (310 mL/min) andneutralzation buffer (265 mL/min). Thus, in both FIGS. 5A and 5B, thecircles represent an air/lysate ratio of 38% (600 ml per min air/1565 mlper min combined lysate flow), the triangles represent an air/liquidratio of 58% (900 ml per min air/1565 ml per min combined lysate flow)and the diamonds represent an air to lysate ratio of 115% (1800 ml permin air/1565 ml per min combined lysate flow).

Comparing the data in each figure, it was found that better recovery wasachieved when air was introduced prior to the first static mixer. Theair delivered through the “T” creates primarily air pockets. The fineair bubbles (<1 mm) required for flotation are thought to be generatedby the turbulent mixing action inside the static mixers. Therefore,flotation is enhanced when air is introduced at the front end of thestatic mixers rather than the middle.

Air Flow Rate Determination: A second set of experiments was designed todetermine the relationship between the liquid and air flow rates forflotation. In this experiment, combinations of three liquid linearvelocities and three air flow rates were tested on the same ⅜″ staticmixer used in the first experiment. Liquid flow rates were maintained byperistaltic pumps at the settings shown in Table 1. Linear velocitiesshown in Table 1 were calculated based on empty cylindrical tubeswithout air introduction. Air flow was controlled by a mass flowcontroller (Cole-Palmer, Model P-33115-60) at 100-500 mL/min.

The best separation occurred at the lowest airflow rate among the threesettings tested. However, the optimal amount of air may be lower thanthis flow rate based on extrapolation of the results. With high air flowrates, the excess air introduced into the static mixers created foamingthat served to increase the total volume of the floating precipitate. Asa result, the recovery of liquid as a percentage was negativelyaffected.

Most of the compaction occurred during the initial hour after celllysis. After 16 hours of settling, small amount of precipitate began tofall to the bottom of the containers. Sinking precipitate can pose aproblem, such as fouling for the subsequent filtration step, indicatingthat unclarified lysate, i.e. where the liquid phase has not beenremoved from the flocculent phase, should not be allowed to standindefinitely.

Determination of Optimal Flow Rates through a “T”: The previous set ofexperiments served to define the effective location of air entering thestatic mixers. The present set of experiments were designed to optimizethe amount of air required for flotation. Since the floating and sinkingbehavior of the precipitates is dependent on the formation of themeta-stable complex between the irregularly shaped cell debris and airbubbles, both the amount of air in the system and the size of the celldebris should be optimized simultaneously to produce the best result.One of the most critical parameters for controlling the size of the celldebris is the linear velocity of the fluid stream. Changes in the ratioand concentration of each buffer should be optimized for efficiency oflysis with optimization of total flow rate where the size of cell debrisis the parameter to be influenced. High linear velocity inside thestatic mixer increases the shear rate, which affects the size of celldebris and air bubbles.

In this set of experiments, a total of ten lysate samples weregenerated, including a control sample with no addition of air. Theselysates were allowed to settle while the upward movement of thelysate-precipitate interface was tracked. After two hours of settling,when the rate of compaction slowed, recoveries of lysates were recorded.FIG. 6 shows the relationship between flotation, linear velocity, andairflow rates.

As demonstrated in FIG. 6, different maximal values of air-to-liquidratio were found for different linear velocities in the static mixers. Atrend of better recovery for high linear velocities at lowerair-to-liquid ratio was observed. The trend may be best explained by thereasoning that air bubbles are sheared to finer size at higher linearvelocity. The drag force per unit volume is inversely proportional tothe diameter of the bubbles. With larger drag force acting against them,smaller air bubbles generated at high linear velocity actually moveslower and stay in the lysate solution longer, which may facilitateformation of the precipitate/air bubble complex necessary for flotation.

EXAMPLE 3

Sparge Stone Development: A set of experiments was run on a ⅝″ i.d.static mixer setup similar to the arrangement shown in FIG. 2 (each ofStatic Mixers 210 and 211 are formed by connecting in series 3individual 12 element mixers: ¾ inch o.d. KENICS ¾ KMR-SAN 12 sanitarymixers obtainable from Chemineer, Inc.). In this experiment, air wasdelivered into the static mixers by means of a stainless steel “spargestone” located at first Air Inlet 5. A schematic diagram of oneembodiment of a sparge stone including optional housing is shown inFIG. 1. Referring to FIG. 1, sparge stone 11 features 2 micron pores toproduce air bubbles less than 1 mm in diameter. During operation, celllysis buffer enters from one port of housing 12 and completely fills theinterior space. Air is controllably introduced through a bottom port ofhousing 12 under pressure and enters the lysate stream through thesparge stone 11 to produce fine bubbles in the lysate stream. Theairflow rate was controlled at 0.25 -1.5 L/min by a mass flowcontroller. Buffer flow rates were set at 1.2, 2.0, 1.0, and 0.85 L/min,respectively, for the four buffers during cell lysis. The compaction offloating precipitates was monitored for up to 2 hours. The sameexperiment was repeated with the “T” instead of the sparge stone forcomparison.

Determination of Optimal Air Flow Rates through “Sparge Stone”: Havingconcluded that air bubbles, especially those with small diameter, seemto be the major determining factor for flotation of precipitates, a moreefficient method of generating the bubbles was sought. In light ofresults from previous experiments, a sparge stone made with stainlesssteel was designed with such intent. The sparge stone, which wasinstalled at the front end of the static mixers, features pores thatgenerate air bubbles of <1 mm in diameter at flow rate of up to 5 L/min.The flow rate was controlled by a mass flow controller placed upstreamof the sparge stone. In this set of experiments, the buffer flow ratesare fixed, producing a linear velocity inside the first mixer of 0.88ft/s. Based on the results from previous experiment using addition ofair through the “T”, the optimal flotation condition was expected to beachieved with airflow rate equivalent to less than 20% of the totalliquid flow rate. The liquid phase recoveries for several air-to-liquidratios are shown in FIG. 7.

As shown in FIG. 7, the optimal condition was found at ˜10%air-to-liquid ratio. Note that the requirement for air is much lowerthan previous experiments where air was introduced through a “T.”Moreover, the extent of compaction of the floating precipitate at agiven time was found to be slightly better than the previousexperiments. In general, by using the sparge stone instead of the “T”,better liquid phase recovery was achieved with less air and lower linearvelocity.

EXAMPLE 4

Effect of Flotation on pDNA Quality: A set of experiments was focused onexamining the effect of the flotation method on the quality of theplasmid. The ⅝″ static mixers were set up with the sparge stone as inthe previous set of experiments. Cell lysis was repeated using threelinear velocities as per Table 2 while air was delivered through thesparge stone at either 12% or 30% of the total flow rate.

TABLE 2 Liquid Flow Rates for ⅝″ Static Mixers Linear Velocity in StaticMixer #1 Flow Rates in mL/min 0.30 ft/s 0.74 ft/s 0.88 ft/s CellRe-suspension 410 1000 1200 Cell Lysis Buffer 680 1670 1990 CellPrecipitation Buffer 340 840 1000 Neutralization Buffer 290 720 850

The lysates were centrifuged after cell lysis and subsequently filteredthrough 1.2 micron GF2 and 0.2 micron ULTIPORE N66 capsules. At thatpoint, the plasmid concentration in each lysate was determined by HPLC.Each clarified lysate was then loaded onto a column packed with TMAEanion exchange resin for purification. Column eluates (purified pDNA)were analyzed for residual host DNA (genomic DNA) concentration. Sampleswere assayed for genomic DNA using a slot blot assay, except that thearrangement of samples and sample dilutions on the blot were changed tomaximize the number of samples assayed on each blot.

Effect of Linear Velocity and Air Flow Rate on the Quality of Plasmids:It is well known that DNA is a delicate macromolecule that isshear-sensitive. The question arose whether the high liquid and air flowrates required for flotation might inflict damage on the pDNA such asconversion from supercoiled to open-circular, or lead to breakage ofgenomic DNA into fragments, which would be difficult to separate fromthe pDNA in subsequent steps. In this set of experiments, alkaline celllysis was run at several combinations of liquid and airflow rates.Plasmid yield for these lysates is shown in FIG. 8.

Plasmid yield showed a declining trend when the linear velocity (in thefirst mixer) increased from 0.3 to 0.88 ft/s. The drop in plasmid yieldmight be explained as insufficient contact time between the cells andthe lysis solution in the first mixer. On the other hand, differences inthe amount of air addition have minimal effect on the plasmid yield.However, if excessive amounts of air were introduced into the staticmixer, efficiency of cell lysis would likely be reduced as air mightprevent contact between the cells and the lysis buffer.

The lysates produced in these experiments were further processed byfiltration and anion exchange column chromatography. Comparisons betweenthe residual endotoxin and chromosomal DNA content in column eluates areshown in FIGS. 9 & 10. As shown in FIGS. 9 and 10, higher residualcontent of both endotoxin and chromosomal DNA were found in the purifiedpDNA from lysates produced at faster flow rates. High linear velocity inthe static mixers translates to higher shear rate for all the componentsin the lysates, and macromolecules are extremely sensitive to shearrate. At higher flow rates, it appears that more residual cell debriswas sheared to comparable size of the plasmid, and became inseparablefrom the pDNA by the subsequent processing steps.

The floating and sinking behavior of cell debris was characterized by amodel based on the change in density of the two phases in the lysate.Fine air bubbles with less than 1 mm in diameter provided superiorflotation of cell debris. It was observed that the flotation of celldebris is affected by the flow rates of both air and buffers. Inaddition, both plasmid yield and quality of lysate were determined todecrease with higher linear velocity in the static mixers. Based on thefindings of this study, the preferred conditions for flotation of celldebris from the lysate were determined to be: linear velocity maintainedat 0.3 ft/s with 12% air introduced through a “sparge stone” deviceplaced in-line with the cell lysis buffer conduit prior to entry of thecell suspension and before a first set of static mixers.

EXAMPLE 5

Fermentation and cell paste: In one embodiment, plasmids having the pUCori are employed in DH5α Escherichia coli (E. coli) hosts. The inoculumis prepared from the Master Cell Bank using a target density of 2-6×10⁷colony forming units per milliliter (“cfu/mL”). The cells are grown in ashake flask under the selective pressure of 50 micrograms/milliliter(micrograms/ml) kanamycin. After reaching 2-6×10⁷ cfu/mL, the inoculumis used to seed the bioreactor, or the material may be held for notlonger than 8 hours at 2-8° C. prior to further processing.

The inoculum is seeded into the bioreactor at a density of >1.0×10⁶colony forming units/milliliter (cfu/ml). Where the plasmid has anantibiotic resistant gene such as for example KanR, the cells are grownunder the selective pressure of 50 micrograms/mL kanamycin.Alternatively, the plasmid copy number may be regulated by conferringother beneficial traits known to those of skill in the art. For example,plasmid copy number may be increased utilizing repressor titration inaccordance with U.S. Pat. No. 6,103,470, incorporated herein byreference.

A polypropyleneoxide-polyethyleneoxide block polymer may be added tocontrol foaming. The pH is controlled by sulfuric acid and sodiumhydroxide. The temperature and dissolved oxygen are also controlled. Thebatch fermentation is halted when the cells exhaust nutrients (typically18 to 22 hours later) and the culture is cooled and harvested.Fermentation end point is based on the off gas CO₂ levels representing aspike in acid & base utilization.

The fermentor culture is cooled to approximately 15° C. and harvestedusing a centrifuge at approximately 15,000 rpm to generate a cell pastethat is 45-50% wet weight. The cell paste is processed immediately, orit may be stored at ≦−60° C. for up to 3 years prior to furtherprocessing. The weight of cell paste determines the volumes of thesubsequent steps.

Cell Resuspension: If frozen, the frozen cell paste was broken by aplastic mallet into pieces of <2×2 centimeters (cm) or pelletized by amill. Broken cell paste was suspended in Resuspension buffer (25 mMTris, 10 mM Na₂EDTA, 50-83 mM dextrose pH 8.50, dextrose is added onlyif the paste has been frozen) at a ratio of 5 liters per kilogram ofcell paste and maintained between 10-25° C. During paste addition, thecell suspension is gently mixed with a bow-tie impeller at approximately200-400 rpm, typically for less than 1 hour although other suitablemeans of mixing can be employed. The optical density of the cellsuspension should be less than or equal to 100 OD₆₀₀ for efficient lysisand recovery of plasmid DNA (“pDNA”). In this example, 5 mL of RNase Aper kg of cell paste (approximately 15,000-24,000 Kunitz units/kg) isadded to digest RNA and the cell suspension is mixed for about 1 to 2hours at ambient temperature using bow-tie impeller prior to in-linelysis.

The OD₆₀₀ is measured. If the OD₆₀₀ is >100, the cell paste solution isdiluted with Resuspension Buffer until the OD₆₀₀ less than or equal to100 and mixed thoroughly for at least 5 minutes after buffer addition.The pH is adjusted to 8.0±0.5 with 2.5 M Tris buffer and mixedthoroughly for at least 5 minutes after buffer addition. The resultingcell suspension is then subject to lysis.

Continuous Flow In-Line Processing: Following cell suspension, theprocess is preferably conducted in a contained continuous flow in-lineserial manner. The in-line apparatus depicted schematically in FIG. 4allows for continuous contained flow through a fluid conduit having aseries of in-line mixers interspersed with input ports, including portsthat permit entry of the cell suspension from source 43, a cell lysisbuffer from source 44, a precipitation buffer from source 47, and,optionally a pH adjustment buffer or neutralization buffer from source48. The cell suspension, cell lysis, precipitation, and, optionally pHadjustment buffers, are each introduced into the in-line system atcontrolled rates using a pump in conjunction with a flow meter in theline leading from each of the sources 43, 44, 47 and 48. When theprocess is primed, the pumps are started essentially simultaneously.Thus, the upstream portion of the system is primed with cell lysisbuffer alone while downstream portions of the system further include theaddition of precipitation and pH adjustment buffers. Once the system isinitiated, it runs continuously until all of the cell suspension hasbeen processed.

Cell Lysis: After priming, the cell suspension is pumped from source 43at a rate of approximately 2 liters per minute (LPM) into the in-linesystem where the cell suspension comes into contact with Cell LysisBuffer (1% SDS w/v, 0.2N NaOH). Cell Lysis Buffer is pumped from source44 at an approximate ratio of 10 liters per kilogram of initial cellpaste and at a rate of approximately 3 LPM. Where SDS:NaOH is employed,the ratio of SDS:NaOH in the lysis solution is controlled to anoptimized ratio of 1% SDS:0.2N NaOH with an acceptable variation of ±10%for comparable plasmid yield. The sequence of initiation of the processis in order: Cell Resuspension pump on, gas inflow on, Cell Lysis Bufferpump on, Precipitation Buffer pump on, and pH Adjustment Buffer pump on.

Introduction of Gas: USP grade nitrogen from source 45 is introducedthrough a sparge stone 41 disposed in housing 42 into the linecommunicating the Cell Lysis Buffer to the in-line system. Air has alsobeen shown to be effective in the process and it is likely that othergases, especially those lighter than air, will work also. Hydrogen andoxygen are less desirable for safety reasons in addition to potentialproblems with oxygen acting as an oxidization agent on the biologicalmaterial.

FIG. 3 provides a schematic depiction of the placement of the gas sourcein the in-line system. As depicted in FIG. 3, the Cell Lysis buffer iscontrollably added from source 34 through a flow meter 1 under theaction of a pump 2. The gas is provided from air/nitrogen source 35through regulator 7 under the control of mass flow regulator 8. Pressurebox 9 provides monitoring of various the input pressures and flow rates.In the embodiment depicted, the gas enters the Cell Lysis Buffer flowthrough a sparge stone 31 fixed in housing 32 through which the CellLysis Buffer flows prior to entry of the Cell Resuspension into the flowpath. The Cell Resuspension is provided from reservoir 33 through a flowmeter 3 under the action of a pump 4. In the embodiment depicted, theflow rate is monitored as the combined flow of the Cell Resuspension andCell Lysis Buffer together with entrained air enters the first of a setof static mixers 310.

FIG. 1 provides a diagram of the sparge stone 11 fixedly placed inhousing 12 in accordance with one embodiment of the invention. In thisembodiment the sparge stone 11 is approximately 3 inches long andapproximately ⅜ of an inch in diameter and is fixed mounted in housing12 by a compression clamp. In this embodiment, the sparge stone 11 isstainless steel and has approximately 2 micron holes through which thenitrogen passes thus forming bubbles of a controlled size uponcontacting the Cell Lysis Buffer. In this embodiment, the sparge stone11 is placed upright in-line in the housing 12. The housing 12 is placedin a line leading from a reservoir holding the Cell Lysis Buffer at apoint just prior to the point at which the Cell Resuspension isintroduced. Nitrogen is added at a rate of 0.99 L/min through the spargestone 11.

Precipitation pH Adjustment and Clarification: As depicted in FIG. 4,the Cell Resuspension and Cell Lysis Buffer containing entrainednitrogen bubbles of defined size flows together in-line through a seriesof in-line static mixers 410 thus effecting mixture with, and lysis of,the cells. The flow rate is approximately 0.3 linear feet per second(ft/sec). Precipitation buffer (3M potassium acetate, 2M acetic acid, pH5.5; at a ratio of ˜5 liters per kilogram of cell paste) from source 47is added in-line to precipitate cell debris, and the mixture continuesto flow through a second set of static mixers 411. pH Adjustment Buffer(2.5 M Tris, 4-5 liters per kilogram of cell paste) is added in-linefrom source 48 to adjust the pH of the lysate, which then flows througha short static mixer 10 before collection into Lysate Clarification Tank52. Each solution is added at a controlled flow rate, with the initiallinear flow rate through the mixers kept at approximately 0.3 ft/sec,which increases to as much as approximately 1.1 ft/sec as furthervolumes are added with addition of the Precipitation and pH AdjustmentBuffers. The flow rate of each solution into the static mixers can bevaried ±10% with no detectable effect on plasmid yield or purity.

The unclarified precipitated and neutralized lysate is allowed to flowgently into the bottom of the Lysate Clarification Tank 52. The lysateis allowed to separate for 1-2 hours at ambient temperature. Thefloating precipitate/flocculent 50 rises to the top of the tank due tothe air bubbles introduced to the mixture and overlies a substantiallyclear fluid representing a clarified lysate. The floating precipitate 50forms quickly but is allowed to rest for a period of 1-2 hours in orderto coalesce and solidify. The underlying clarified lysate 51 is removedfrom the bottom of tank 52 either by pumping through a dip tube 54 or,optionally, by draining through a bottom port 53.

The clarified lysate 51 obtained as the clear liquid underlying thefloating flocculent is further purified by filtration a series offilters 55 to generate a filtered lysate that can be subject to furtherpurification if desired. In one embodiment the filter series 55 is afollows: a 8 micron depth filter (Seitz Bio-40, 170 L/m² capacity), a 2micron depth filter (Seitz Bio-10, 170 L/m² capacity), a 1.2 micronpre-filter (1.2 micron glass fiber absolute filter, Sartorius, GF2, 5L/ft² capacity) and 0.2 micron nominal filter (0.2 micron nylon absolutefilter, Sartorius or Pall-Gelman N₆₆, 5 L/ft² capacity) in series. Theflow rate is 0.2 L/min/ft², based on the area of the smaller of thepre-filter or the 0.2 micron filter, using a peristaltic pump, capableof 0.2 L/min/ft² of 0.2 micron filter at 10 psig. Lysate capacity of theprefilter and 0.2 micron filter is 5 L/ft². Filtration is continueduntil the introduction of precipitate causes a pressure of >10 psi overthe Bio-40, at which time that filter is removed from the train and theremaining lysate in the filter housings is recovered.

Alternatively, the clarified lysate can be filtered through a Miracloth.The pH and conductivity of the Miracloth filtered lysate is measured. Ifthe filtered lysate pH is below 8.0, it is adjusted with 2.5 M Trisbuffer. If the conductivity is greater than 60 mS/cm, it is adjustedwith purified water. The Miracloth filtered lysate is pumped through a1.2 micron Glass fiber (GF2) filter in tandem with a 0.2 micron Nylonfilter. The volume of Miracloth filtered lysate that can go through bothfilters is 5 liter per ft² per filter. The pump flow rate through thefilter is 0.2 liter per minute per ft² with a pressure drop no greaterthan 10 psi per filter.

The filtered lysate may be held at ambient temperature for 24 hours orat 2-8° C. for up to 1 week prior to initiating the next process step.Filtered product cannot be frozen because of precipitate formation oraggregation of small particle size upon thawing. These requirere-filtration of the product before processing can continue. Anionexchange HPLC chromatography is used to estimate the plasmid DNAconcentration in process samples. A peak area standard curve delineatesan approximation of the amount of supercoiled DNA and open circular DNApresent in the sample.

Anion Exchange Chromatography: An anion exchange resin,Trimethylaminoethyl Fractogel 650M (Merck/EM Separations) is used toseparate intact plasmid DNA from plasmid impurities and host impuritiessuch as residual protein, endotoxin, chromosomal DNA and digested RNA.The column is equilibrated with an Equilibration Buffer: 50 mM Tris,0.55 M NaCl, pH 8.5. Most of the impurities do not bind to the resin orare removed from the column by sequential washes of increasing saltconcentration, starting with the Equilibration Buffer. More tightlybound components are washed off with a buffer of higher saltconcentration [50 mM Tris, 0.65 M NaCl, pH 8.5]. The plasmid DNA iseluted from the column with a yet higher salt concentration [50 mM Tris,0.71 M NaCl, pH 8.5]. The eluent is filtered through a 1.2 micron glassfiber filter (Sartorius, 1 ft₂ membrane per g pDNA) and then a 0.2micron nylon filter, (Sartorius or Pall-Gelman N₆₆, 1 ft² membrane per gpDNA), into an irradiated container. The column eluent may be held forno longer than 1 week at 2-8° C. prior to further processing. The exactsalt concentration used for the above buffers is established duringresin acceptance testing of each lot of resin to compensate for thevariation in charge density of the resin from lot to lot. Theconcentration typically varies ±0.05M NaCl.

Ultrafiltration/Diafiltration: Ultrafiltration and diafiltration areused to concentrate, clear small molecular weight components, and toexchange the ion exchange column eluent into a 10 mM Tris, pH 8.0 bufferfor formulation. A description of the use of ultrafiltration inconjunction with in-line lysis and ion exchange chromatography isprovided in Dang, Bussey and Bridenbaugh, WO 00/05358, incorporatedherein by reference. In the present embodiment, ultrafiltration isemployed following ion exchange chromatography. Typicallyultrafiltration is carried out essentially as described in Bussey etal., U.S. Pat. No. 6,011,148, incorporated herein by reference.

Ultrafiltration (UF): A open screen tangential flow ultrafiltrationcassette is used together with a modified polyethersulfone based, 50,000Dalton molecular weight cutoff ultrafiltration membrane chosen for itscapacity to retain plasmid DNA and pass small molecules. The filteredion exchange eluate is connected to the UF feed line. The UF/DF systemis operated at 0.5-1.0 L per minute per ft² with a TMP of 2-4 psig. Whennecessary, the flow rate is lowered to maintain a TMP of 2-4 psig. Thepermeate valve is opened and the permeate line is directed into thefiltered ion exchange pool. This material is recirculated through thesystem to build a gel layer for 20-30 minutes at TMP 2-4 psig. A gellayer is built when permeate UV_(260 nm) is less than 0.1 mg/ml DNAthrough the permeate line. Without stopping the pump, the permeate lineis directed to drain and the plasmid DNA is concentrated until aconcentration of ≧3 mg/mL is achieved before proceeding to thediafiltration step.

Diafiltration (DF): Without shutting off the pump, diafiltration buffer(10 mM Tris, pH 8.0) is fed into the recirculation tank. The plasmid DNAis concentrated, diafiltered to achieve a conductivity of less than 0.8mS/cm at 25° C. or equal to the diafiltration buffer conductivity(typically 8 to 12 DF volumes) and then concentrated to >2 mg/ml, asnecessary. The diafiltered concentrate is immediately processed. Ifnecessary, ultrafiltration may be continued until a target concentrationof ≧3 mg/mL is achieved.

Final Filtration: The UF/DF plasmid DNA Pool is filtered through a 0.2micron filter at 0.01 to 0.02 mL/min/cm² into a sterile container.

The examples and embodiments described herein are for illustrativepurposes only, and various modifications will be apparent to those ofskill in the art, the invention to be limited only by the scope of theappended claims. All publications, patents and patent applications citedherein are hereby incorporated by reference as if set forth in theirentirety herein.

1. A method of producing a clarified cell lysate comprising plasmid DNAfrom an alkaline bacterial cell lysate, comprising the steps of:introducing a suspension of bacterial cells into a fluid flow comprisingan alkaline lysis buffer and an entrainment of gas, wherein the cellsare flowably mixed with the cell lysis buffer together with the gasthereby forming a cell lysis mixture; introducing a precipitation bufferinto the fluid flow comprising the cell lysis mixture, thereby forming aprecipitated lysate; introducing a pH adjustment buffer into theprecipitated lysate and combining the pH adjustment buffer and theprecipitated lysate prior to separating the mixture; separating themixture into a buoyant flocculent phase comprising precipitated celldebris and a fluid phase comprising a substantially clarified celllysate; and isolating the clarified cell lysate.
 2. A method ofproducing a clarified cell lysate comprising plasmid DNA from analkaline bacterial cell lysate, comprising the steps of: introducing asuspension of bacterial cells into a fluid flow comprising an alkalinelysis buffer and an entrainment of gas, wherein the cells are flowablymixed with the cell lysis buffer together with the gas thereby forming acell lysis mixture; introducing a precipitation buffer into the fluidflow comprising the cell lysis mixture, thereby forming a cell debrisprecipitate in the cell lysis mixture; introducing the cell lysismixture including the cell debris precipitate into a lysate separationtank and separating the mixture into a buoyant flocculent phasecomprising the precipitated cell debris and a fluid phase comprising aclarified cell lysate, and isolating the substantially clarified celllysate.
 3. A method of producing a clarified cell lysate comprisingplasmid DNA from an alkaline bacterial cell lysate, comprising the stepsof: introducing a suspension of bacterial cells into a fluid flowcomprising an alkaline lysis buffer and an entrainment of gas andflowably mixing the cells with the cell lysis buffer together with thegas by passage through a first static mixer, thereby forming a celllysis mixture; introducing a precipitation buffer into the fluid flowcomprising the cell lysis mixture and flowably mixing the cell lysismixture with the precipitation buffer by passage through a second staticmixer, thereby forming a cell debris precipitate in the cell lysismixture; introducing a pH adjustment buffer into the fluid flowcomprising the cell debris precipitate and flowably mixing the celllysis mixture with the pH adjustment buffer by passage through a thirdstatic mixer, thereby forming a pH adjusted cell lysis mixture; flowingthe pH adjusted cell lysis mixture into a lysate separation tank forseparating the cell lysis mixture into a buoyant flocculent phasecomprising the precipitated cell debris and a fluid phase comprising asubstantially clarified cell lysate; obtaining the substantiallyclarified cell lysate from under the buoyant flocculent phase; andfiltering the substantially clarified cell lysate to form a clarifiedcell lysate.
 4. The method of claim 3, wherein the gas is introduced viaa gas port through which a gas is forced under pressure into the fluidflow thereby controllably forming bubbles in the cell lysis mixture. 5.The method of claim 4, wherein the gas port comprises an aperturecomprising a plurality of pores.
 6. The method of claim 5, wherein thepores have an average diameter of less than 5 microns.
 7. The method ofclaim 5, wherein the aperture comprising a plurality of pores is asparge stone or disk filter comprising pores having an average diameterof 2 microns or less.